The invention relates to a microreactor array, a device comprising a microreactor array, and a method for using a microreactor array. By means, of conventional titer plates it becomes possible to implement microreactor arrays of, for example, 6, 24, 48, 96, 384 or more individual microreactors. Just as the number of microreactors varies greatly, so can the volume of the individual reactors. While the term “microreactors” is used already at scales of below 10 ml, further reduction of the volume to below 1 ml, below 500 μl, below 100 μl or even below 10 μl can be associated with some advantages. The surface-to-volume ratio that has been increased by the volume reduction is larger, and oxygen input into the reaction solution through simple diffusion is simpler. The microreactor arrays can be shaken continuously until the end of the reaction. Individual metered addition makes it possible to carry out various experiments in each individual reactor. The invention is, in particular, also suitable for automating screening experiments in a fedbatch mode, a continuous mode and/or pH regulation. The invention relates in particular to microbial, biochemical, enzymatic and chemical reactions. The invention supports sterile, aseptic or monoseptic operation.
Process parameters such as for example the pH value, concentrations of dissolved oxygen, dissolved carbon dioxide, biomass, educt and product, or temperature can be used as controller outputs for controlling the metered addition. Particularly suitable, in the context of the invention, is a non-invasive acquisition of the process parameters with the use of optical or electromagnetic measuring methods through the bottom that is permeable to electromagnetic radiation.
In many areas of biology, process technology, pharmacy and medicine, screening of biological systems becomes necessary (e.g. selection of suitable biological strains, enzymes or suitable culture media and culture conditions). In this context there is a need for high sample throughputs (parallelisation of the experiments) and for a reduction in the quantity of reactants, some of which are expensive.
By means of the bioreactors in use at present, for example shaker flasks, small-scale fermenters and test tubes, it is not possible to meet this demand. Established techniques do not meet the requirements for automation, cost minimisation and the required high throughput. In particular in the case of biocatalytic systems, there is a requirement to carry out many parallel experiments at the microliter scale, because such processes, generally speaking, progress more slowly and especially in the development phase are more expensive than comparable chemical processes. It is therefore necessary to develop microbioreactors that in the smallest possible space provide a suitable environment for biological cultivation and biocatalytic reactions. In this context two criteria should be underlined as being important prerequisites for suitable operating conditions: the possibility of carrying out the corresponding experiments under sterile or monoseptic conditions, and ensuring mass transfer (liquid-liquid; liquid-gas; solid-liquid; solid-gas) that is suitable and adequate for the biological culture or for the biocatalytic reaction system.
Microreactor arrays, for example microtiter plates (MTPs) provide an ideal platform in order to achieve a high number of parallel operations. Due to the small reaction volumes (e.g. <10 μL to >5 mL per chamber), the high number of parallel operations (e.g. 6 to 1536 chambers per plate) and the option of automating the cultivation processes (in a form that can be handled by robots), microreactor arrays represent the bioreactor that overall is most cost-effective and that has the most promising future. Furthermore, the use of non-invasive optical measuring methods in order to acquire process variables is already quite advanced in this reactor type. Moreover, the operating conditions in shaken MTPs as far as the mass transfer (maximum oxygen transfer capacity) is concerned are already well characterised (Hermann et al., 2002) and are thus comparable to the operating conditions in laboratory reactors, pilot reactors or production reactors.
For these very reasons there has recently been an increase in the use of microreactor arrays. At present, microtiter plates are already used for screening biological systems. To this effect the individual reaction chambers are filled, inoculated, and incubated on a rotary shaker. As a result of the shaking movement the feed of oxygen into the reaction fluids is improved, and thorough mixing of the reaction fluids is achieved. Apart from the shaking movement there are other possible options that may be considered for thoroughly mixing fluids in microtiter plates (e.g. moving the fluid by means of sound waves). In order to keep the system sterile, the microtiter plates are covered by an air-permeable membrane (pore size <0.2 μm) or airproof film or foil or a cover construction, or cultivation takes place in an open manner in a sterile environment.
In order to carry out the different biocatalytic reactions it is often necessary to add to the ongoing reaction various fluids or gasses while the experiment is ongoing. In this context the supply of pH adjusters (alkaline solutions and acids) for pH titration of the ongoing reaction, and the infeed of substrates should be emphasized. It is only the infeed of substrates during the process that makes it possible to carry out regulated batch, fedbatch and continuous processes. These modes of operation are particularly important for flexible and successful biological screening and for further development of the processes.
A brief discussion of the importance of the most important operating methods is provided below.
The pH value of a medium is one of the most important environmental influences for cell growth and for the ability of micro-organisms to develop. The activities of the enzymes that catalyse all the reactions due to the metabolic process are decisively influenced by the pH value. However, the pH value of the medium continuously changes as a result of the metabolism of the organisms and as a result of the consumption of the components of the culture medium. If pH regulation is lacking, it is difficult to achieve high cell densities. Therefore regulation of the pH value by titration of pH adjusters (alkaline solution or acid) is necessary. The pH adjuster can be in liquid or gaseous form.
In industrial production processes the fedbatch mode (cultivation with the infeed of substrate) is the standard process. The fedbatch mode is necessary in many production processes, for example in order to prevent inhibition as a result of excessive substrate concentrations or catabolite repression. Fedbatch mode must therefore be possible for the corresponding bioreactors. In addition it is precisely in this mode, where the pH value changes greatly, that pH regulation is almost always necessary.
Furthermore, continuous reaction management of biocatalytic systems is important in order to achieve a steady state or steady-state balance and in order to determine kinetic parameters, as well as to carry out long-term experiments (e.g. to determine the genetic stability of genetically modified micro-organisms).
For many decades, regulating the pH value and carrying out the reactions in fedbatch mode has been the state of the art in large-scale industrial applications. Likewise, commercially available small-scale (>10 mL-500 mL) reactor systems are becoming increasingly widespread (e.g. fedbatch pro by DasGip [U.S. Pat. No. 6,202,713], Sixfors by Infors, etc.).
Small-scale reactor systems are used for screening and process development; they can be operated under the above-mentioned conditions. However, the following applies: the smaller the reaction volume, the more elaborate and cost-intensive the implemented solution. Due to the very considerable technical expenditure in the case of small reaction volumes, and due to the low achievable degree of parallel operation in the case of greater reaction volumes, the proposed approaches to this problem are associated with very considerable limitations. For example, in the current approaches it is not possible to have high-throughput screening (>16, >20, >50, >500, >1,000, >10,000 to >100,000 reactions per day) while at the same time providing high-content screening (>1, >2, >3, >4, >5 reaction parameters for each reaction point in time). At present different approaches to finding a solution have been implemented or are pursued to implement the described operational modes in microreactor arrays. Broadly-speaking, a differentiation can be made among contactless, contacting, and microfluidic dosing methods.
The technique for contactless metered addition of very small quantities of fluid is largely based on piezo technology. As a result of the very considerable instances of acceleration (up to 100,000 g) that can be achieved by a piezo element, it is possible to separate, and thus to dose, very small droplets (>30 pL) from a reservoir. Various companies (e.g. Nano-Plotter™ by GeSim; Autodrop by Microdrop) market devices for the metered addition of very small volumes of fluid in MTPs.
Furthermore, contactless dosing methods exist that are operated with the use of compressed air (e.g. Vieweg GmbH) [EP 1036594]. The achievable droplet volumes range from approximately 5 μL to 0.2 mL. Furthermore, there are patents relating to the cultivation of cells in microreactors with the use of droplet infeed of reaction fluids [DE 10221565, DE 10019862, DE 10046175]. In these approaches the reaction fluids are fed through capillaries to the reaction chamber or above the reaction chamber until at the tip of the capillary a droplet of adequate size has formed, which droplet then drips into the reaction space. In the case of aqueous media the volume of such a droplet is in the order of approximately 20 μL, depending on the diameter and the design of the needles.
In contacting dosing methods the infeed of fluids mostly takes place by controlled dipping of pipette tips into the reaction fluids of the individual chambers of the microreactor arrays by way of a pipetting robot. In this process serially (one pipette tip) or in parallel (several pipette tips) the desired quantities of fluids are metered to the individual chambers. By dipping the pipette into the reaction fluid the metered droplet can be separated from the pipette tip so that quantities in the picoliter range can also be dosed [DE 10201749, DE10116642, WO 02/080822].
Utilisation of microfluidic transport in microreactors is not new either. There are already several approaches for transporting and controlling minute quantities of fluid in microreactors, e.g. WO 02/37096, U.S. Pat. No. 6,403,338, EP 1142641, DE 60000109, U.S. Pat. No. 6,268,219, WO 01/68257, US 2005/0032204; US 2005/0084421, WO 02/060582, WO 03/025113, WO01/68257, WO2004/069983.
Taking into consideration the current approaches and evaluation of them by applying the criteria of sterile operation of the reaction space, oxygen supply of the biological cultures, and realisation of individual infeed and discharge of fluids, the individual approaches are associated with the following disadvantages.
While contactless jet-dosing based on piezo technology makes it possible to dose very small quantities of fluid, these methods are however cost-intensive and prone to malfunction.
When compared to piezo-technology-based methods, pressure-driven or gravity-driven methods are more cost-effective, but they are associated with a disadvantage in that only comparatively larger droplet volumes (>5 μl or <0.2 mL) can be dosed. As a result of the volume ratio of the infed droplets to the reaction volume of the individual microtiter plates, at the time of adding a droplet of reaction fluid an extreme increase in the corresponding concentrations in the reaction chambers takes place. As a result of these extreme concentration increases the experiments are considerably influenced so that the analysis results obtained are not representative and cannot be transposed to larger scales. Such extreme increases in concentration can then only be counteracted by an increase in the volume of the culture.
Contactless dosing methods are associated with a further disadvantage in that closing off the microreactor arrays by means of a membrane or a cover is either not possible at all or is possible only with very considerable expenditure. Closing them is necessary in order to keep the individual reaction vessels of an MTP sterile or monoseptic, and in order to minimise any evaporation of reaction fluid.
Furthermore, contactless dosing methods do not allow dosing during continuous shaking of microreactor arrays. Only intermittent dosing is possible, because any metered addition is only possible after the shaking process has been stopped. However, in the case of biocatalytic reactions non-intermittent metered addition is desirable because as a result of stopping the shaking movement the operating conditions (e.g. interruption of the oxygen infeed and of thorough mixing) undergo a sudden change, and thus any analysis results obtained in this manner can be used only to a limited extent.
In the case of contacting dosing methods there is a considerable danger of contamination or cross contamination as a result of the pipette dipping into the reaction fluid. Furthermore, with this method, too, closing the microreactor arrays off by means of a membrane or a cover is prevented, and any metered addition of fluids during the shaking process is likewise not possible.
Existing microfluidic approaches relating to the transport and control of minute quantities of fluid in microreactors comprise at least one of the following deficiencies: The methods are not designed to dose variable volumes to individual chambers of microreactor arrays. The methods are exclusively used for the analysis of substances and substance flows, for the separation of substance flows and/or for the preparation of the smallest possible sample volumes. The methods only support even separation of fluid volumes in microreactor arrays. With these methods it is not possible to achieve any targeted metered addition to individual chambers. The methods do not allow any sterile closing off of the microreactor arrays by means of a membrane or a cover. The methods do not support the metered addition or discharge of fluids during continuous shaking of the microreactor arrays in order to thoroughly mix the reaction fluids and in order to set adequate mass transfer rates.
In summary it can be stated that none of the approaches mentioned above make it possible to unify the following requirements of a biotechnological, biochemical or chemical process:    1. individual infeed and discharge of fluids into and from the individual reaction chambers of a microreactor array (e.g. in order to implement pH titration or a fedbatch process);    2. sterile or monoseptic closing-off of the microreactor array during infeed or discharge of fluids;    3. adequate and controlled mass transfer (e.g. for the supply of oxygen) by means of shaking.